Enteroendocrine cells (EECs) are one of five epithelial cell lineages in the intestine that arise from a common intestinal stem cell. Similar to enterocytes and goblet cells, enteroendocrine cells turnover every 3-5 days. The self-renewal of EECs from intestinal stem cells is of considerable importance given the requirement of gut hormones for postnatal growth and survival as well as their roles in targeting the brain and pancreas to regulate food intake and enhance insulin secretion. The homeostatic mechanisms that limit EECs to approximately 1-2% of the intestinal epithelium are not known but probably involve activating Notch in neighboring cells. Notch favors the differentiation of absorptive enterocytes while selecting against secretory lineage cell differentiation by inhibiting expression of basic helix loop helix (bHLH) transcription factors. However, it is not know how Notch specifically limits EEC differentiation as opposed to generally reducing secretory lineage cell fate. The bHLH protein Neurogenin3 (Neurog3) is required for the earliest stages of EEC specification. However, Neurog3+ cells can give rise to other intestinal cell types. Expression of NeuroD, another bHLH protein activated by Neurog3, is restricted to cells that adopt an endocrine cell fate. The three aims of the proposal will examine how the cell fate of Neurog3+ cells is regulated to maintain EEC homeostasis. Aim 1 will characterize the effects of Neurogenin3 gene dosage and expression level on EEC cell fate using mice with one or two Neurog3 mutant alleles to reduce Neurog3 protein expression combined with cell lineage tracing of Neurog3 cells. We expect that reduced Neurog3 protein expression will increase alternate nonendocrine cell fates by Neurog3+ cells, including reversion to pluripotent intestinal stem cells. Aim 2 will examine the effects of Neurog3 gene dosage on Notch pathway activity in EECs and will examine the functional role of the Notch ligand, Jagged2 (Jag2), which is restricted to EECs, in inhibiting neighboring cells from becoming EECs. Studies proposed in Aim 3 will explore the role of NeuroD as a transcriptional effector of Neurog3. We will conditionally delete NeuroD from Neurog3+ cells to determine if it potentiates reduced EEC differentiation seen with reduced Neurog3 expression. We will then determine if conditional expression of NeuroD in Neurog3+ cells rescues the impaired EEC differentiation in Neurog3-/- mice and whether EEC differentiation is increased by conditional NeuroD expression in Neurog3 expressing cells. Finally, NeuroD and activated Notch will both be conditionally expressed in Neurog3+ cells to determine if the potent inhibition of EEC cell fate by Notch is rescued by NeuroD, implying that the effects of Notch are result from loss of NeuroD. Completion of the proposed studies will expand our knowledge of how less abundant intestinal cell types are maintained in the correct proportions.